Azabicyclo-octane inhibitors of iap

ABSTRACT

The invention provides novel inhibitors of IAP that are useful as a therapeutic agents for treating malignancies where the compounds have the general formula I: 
     
       
         
         
             
             
         
       
     
     X 1  and X 2  are independently O or S; L is a bond or —C(X 3 )—, —C(X 3 )NR 12 , —C(X 3 )O— wherein X 3  is O or S and R 12  is H or R 1 ; R 1  is alkyl, a carbocycle, carbocycle-substituted alkyl, a heterocycle or heterocycle-substituted alkyl, wherein each is optionally substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, haloalkyl, alkoxy, alkylsulfonyl, amino, nitro, aryl and heteroaryl; R 2  is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a heterocycle or heterocyclylalkyl; R 3  is H or alkyl; R 4  and R 4′  are independently H, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroaralkyl wherein each is optionally substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino and nitro; R 5 , and R 5′  are each independently H or alkyl; R 6  is H or alkyl; and salts and solvates thereof.

RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 11/088,008, filed Mar. 22, 2005, which claims thebenefit of U.S. Provisional Application Ser. No. 60/555,755, filed Mar.23, 2004. U.S. application Ser. No. 11/088,008 and U.S. Provisional Ser.No. 60/555,755 are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to organic compounds useful for therapyand/or prophylaxis in a mammal, and in particular to azabicyclo-octaneinhibitors of IAP proteins useful for treating cancers.

BACKGROUND OF THE INVENTION

Apoptosis or programmed cell death is a genetically and biochemicallyregulated mechanism that plays an important role in development andhomeostasis in invertebrates as well as vertebrates. Aberrancies inapoptosis that lead to premature cell death have been linked to avariety of developmental disorders. Deficiencies in apoptosis thatresult in the lack of cell death have been linked to cancer and chronicviral infections (Thompson et al., (1995) Science 267, 1456-1462).

One of the key effector molecules in apoptosis are the caspases(cysteine containing aspartate specific proteases). Caspases are strongproteases, cleaving after aspartic acid residues and once activated,digest vital cell proteins from within the cell. Since caspases are suchstrong proteases, tight control of this family of proteins is necessaryto prevent premature cell death. In general, caspases are synthesized aslargely inactive zymogens that require proteolytic processing in orderto be active. This proteolytic processing is only one of the ways inwhich caspases are regulated. The second mechanism is through a familyof proteins that bind and inhibit caspases.

A family of molecules that inhibit caspases are the Inhibitors ofApoptosis (IAP) (Deveraux et al., J Clin Immunol (1999), 19:388-398).IAPs were originally discovered in baculovirus by their functionalability to substitute for P35 protein, an anti-apoptotic gene (Crook etal. (1993) J Virology 67, 2168-2174). IAPs have been described inorganisms ranging from Drosophila to human. Regardless of their origin,structurally, IAPs comprise one to three Baculovirus IAP repeat (BIR)domains, and most of them also possess a carboxyl-terminal RING fingermotif. The BIR domain itself is a zinc binding domain of about 70residues comprising 4 alpha-helices and 3 beta strands, with cysteineand histidine residues that coordinate the zinc ion (Hinds et al.,(1999) Nat. Struct. Biol. 6, 648-651). It is the BIR domain that isbelieved to cause the anti-apoptotic effect by inhibiting the caspasesand thus inhibiting apoptosis. As an example, human X-chromosome linkedIAP (XIAP) inhibits caspase 3, caspase 7 and the Apaf-1-cytochrome Cmediated activation of caspase 9 (Deveraux et al., (1998) EMBO J. 17,2215-2223). Caspases 3 and 7 are inhibited by the BIR2 domain of XIAP,while the BIR3 domain of XIAP is responsible for the inhibition ofcaspase 9 activity. XIAP is expressed ubiquitously in most adult andfetal tissues (Liston et al, Nature, 1996, 379(6563):349), and isoverexpressed in a number of tumor cell lines of the NCI 60 cell linepanel (Fong et al, Genomics, 2000, 70:113; Tamm et al, Clin. Cancer Res.2000, 6(5):1796). Overexpression of XIAP in tumor cells has beendemonstrated to confer protection against a variety of pro-apoptoticstimuli and promotes resistance to chemotherapy (LaCasse et al,Oncogene, 1998, 17(25):3247). Consistent with this, a strong correlationbetween XIAP protein levels and survival has been demonstrated forpatients with acute myelogenous leukemia (Tamm et al, supra).Down-regulation of XIAP expression by antisense oligonucleotides hasbeen shown to sensitize tumor cells to death induced by a wide range ofpro-apoptotic agents, both in vitro and in vivo (Sasaki et al, CancerRes., 2000, 60(20):5659; Lin et al, Biochem J., 2001, 353:299; Hu et al,Clin. Cancer Res., 2003, 9(7):2826). Smac/DIABLO-derived peptides havealso been demonstrated to sensitize a number of different tumor celllines to apoptosis induced by a variety of pro-apoptotic drugs (Arnt etal, J. Biol. Chem., 2002, 277(46):44236; Fulda et al, Nature Med., 2002,8(8):808; Guo et al, Blood, 2002, 99(9):3419; Vucic et al, J. Biol.Chem., 2002, 277(14):12275; Yang et al, Cancer Res., 2003, 63(4):831).

Melanoma IAP (ML-IAP) is an IAP not detectable in most normal adulttissues but is strongly upregulated in melanoma (Vucic et al., (2000)Current Bio 10:1359-1366). Determination of protein structuredemonstrated significant homology of the ML-IAP BIR and RING fingerdomains to corresponding domains present in human XIAP, C-IAP1 andC-IAP2. The BIR domain of ML-IAP appears to have the most similaritiesto the BIR2 and BIR3 of XIAP, C-IAP1 and C-IAP2, and appears to beresponsible for the inhibition of apoptosis, as determined by deletionalanalysis. Furthermore, Vucic et al., demonstrated that ML-IAP couldinhibit chemotherapeutic agent induced apoptosis. Agents such asadriamycin and 4-tertiary butylphenol (4-TBP) were tested in a cellculture system of melanomas overexpressing ML-IAP and thechemotherapeutic agents were significantly less effective in killing thecells when compared to a normal melanocyte control. The mechanism bywhich ML-IAP produces an anti-apoptotic activity is through inhibitionof caspase 3, 7 and 9. ML-IAP did not effectively inhibit caspases 1, 2,6, or 8.

Since apoptosis is a strictly controlled pathway with multipleinteracting factors, the discovery that IAPs themselves are regulatedwas not unusual. In the fruit fly Drosophila, the Reaper (rpr), HeadInvolution Defective (hid) and GRIM proteins physically interact withand inhibit the anti-apoptotic activity of the Drosophila family ofIAPs. In the mammal, the proteins SMAC/DIABLO act to block the IAPs andallow apoptosis to proceed. It was shown that during normal apoptosis,SMAC is processed into an active form and is released from themitochondria into the cytoplasm where it physically binds to IAPs andprevents the IAP from binding to a caspase. This inhibition of the IAPallows the caspase to remain active and thus proceed with apoptosis.Interestingly, sequence homology between the IAP inhibitors shows thatthere is a four amino acid motif in the N-terminus of the processed,active proteins. This tetrapeptide appears to bind into a hydrophobicpocket in the BIR domain and disrupts the BIR domain binding to caspases(Chai et al., (2000) Nature 406:855-862, Liu et al., (2000) Nature408:1004-1008, Wu et al., (2000) Nature 408 1008-1012).

SUMMARY OF THE INVENTION

In one aspect of the present invention there is provided novelinhibitors IAP having the general formula (I)

wherein

-   X₁ and X₂ are independently O or S;-   L is a bond, —C(X₃)—, —C(X₃)NR₁₂ or —C(X₃)O— wherein X₃ is O or S    and R₁₂ is H or R₁;-   R₁ is alkyl, a carbocycle, carbocycle-substituted alkyl, a    heterocycle or heterocycle-substituted alkyl, wherein each is    optionally substituted with halogen, hydroxyl, mercapto, carboxyl,    alkyl, haloalkyl, alkoxy, alkylsulfonyl, amino, nitro, aryl and    heteroaryl;-   R₂ is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a    heterocycle or heterocyclylalkyl;-   R₃ is H or alkyl;-   R₄ and R_(4′) are independently H, alkyl, aryl, aralkyl, cycloalkyl,    cycloalkylalkyl, heteroaryl, or heteroaralkyl wherein each is    optionally substituted with halogen, hydroxyl, mercapto, carboxyl,    alkyl, alkoxy, amino and nitro;-   R₅ and R_(5′) are each independently H or alkyl;-   R₆ is H or alkyl;    and salts and solvates thereof.

In another aspect of the invention, there are provided compositionscomprising compounds of formula I and a carrier, diluent or excipient.

In another aspect of the invention, there is provided a method ofinducing apoptosis in a cell comprising introducing into said cell acompound of formula I.

In another aspect of the invention, there is provided a method ofsensitizing a cell to an apoptotic signal comprising introducing intosaid cell a compound of formula I.

In another aspect of the invention, there is provided a method forinhibiting the binding of an IAP protein to a caspase protein comprisingcontacting said IAP protein with a compound of formula I.

In another aspect of the invention, there is provided a method fortreating a disease or condition associated with the overexpression of anIAP in a mammal, comprising administering to said mammal an effectiveamount of a compound of formula I.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

“Alkyl” means a branched or unbranched, saturated or unsaturated (i.e.alkenyl, alkynyl) aliphatic hydrocarbon group, having up to 12 carbonatoms unless otherwise specified. When used as part of another term, forexample “alkylamino”, the alkyl portion may be a saturated hydrocarbonchain, however also includes unsaturated hydrocarbon carbon chains suchas “alkenylamino” and “alkynylamino. Examples of particular alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl,n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, n-heptyl, 3-heptyl,2-methylhexyl, and the like. The terms “lower alkyl” “C₁-C₄ alkyl” and“alkyl of 1 to 4 carbon atoms” are synonymous and used interchangeablyto mean methyl, ethyl, 1-propyl, isopropyl, cyclopropyl, 1-butyl,sec-butyl or t-butyl. Unless specified, substituted, alkyl groups maycontain one, two, three or four substituents which may be the same ordifferent. Examples of the above substituted alkyl groups include, butare not limited to; cyanomethyl, nitromethyl, hydroxymethyl,trityloxymethyl, propionyloxymethyl, aminomethyl, carboxymethyl,carboxyethyl, carboxypropyl, alkyloxycarbonylmethyl,allyloxycarbonylaminomethyl, carbamoyloxymethyl, methoxymethyl,ethoxymethyl, t-butoxymethyl, acetoxymethyl, chloromethyl, bromomethyl,iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl),2-amino(iso-propyl), 2-carbamoyloxyethyl and the like. The alkyl groupmay also be substituted with a carbocycle group. Examples includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, andcyclohexylmethyl groups, as well as the corresponding -ethyl, -propyl,-butyl, -pentyl, -hexyl groups, etc. Particular substituted alkyls aresubstituted methyls e.g. a methyl group substituted by the samesubstituents as the “substituted C_(n)-C_(m) alkyl” group. Examples ofthe substituted methyl group include groups such as hydroxymethyl,protected hydroxymethyl (e.g. tetrahydropyranyloxymethyl),acetoxymethyl, carbamoyloxymethyl, trifluoromethyl, chloromethyl,carboxymethyl, bromomethyl and iodomethyl.

“Amidine” denotes the group —C(NH)—NHR wherein R is H or alkyl oraralkyl. A particular amidine is the group —NH—C(NH)—NH₂.

“Amino” denotes primary (i.e. —NH₂), secondary (i.e. —NRH) and tertiary(i.e. —NRR) amines. Particular secondary and tertiary amines arealkylamine, dialkylamine, arylamine, diarylamine, aralkylamine anddiaralkylamine. Particular secondary and tertiary amines aremethylamine, ethylamine, propylamine, isopropylamine, phenylamine,benzylamine dimethylamine, diethylamine, dipropylamine anddiisopropylamine.

“Amino-protecting group” as used herein refers to a derivative of thegroups commonly employed to block or protect an amino group whilereactions are carried out on other functional groups on the compound.Examples of such protecting groups include carbamates, amides, alkyl andaryl groups, imines, as well as many N-heteroatom derivatives which canbe removed to regenerate the desired amine group. Particular aminoprotecting groups are Boc, Fmoc and Cbz. Further examples of thesegroups are found in T. W. Greene and P. G. M. Wuts, “Protective Groupsin Organic Synthesis”, 2^(nd) ed., John Wiley & Sons, Inc., New York,N.Y., 1991, chapter 7; E. Haslam, “Protective Groups in OrganicChemistry”, J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973,Chapter 5, and T. W. Greene, “Protective Groups in Organic Synthesis”,John Wiley and Sons, New York, N.Y., 1981. The term “protected amino”refers to an amino group substituted with one of the aboveamino-protecting groups.

“Aryl” when used alone or as part of another term means a carbocyclicaromatic group whether or not fused having the number of carbon atomsdesignated or if no number is designated, up to 14 carbon atoms.Particular aryl groups include phenyl, naphthyl, biphenyl,phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook ofChemistry (Dean, J. A., ed) 13^(th) ed. Table 7-2 [1985]). In aparticular embodiment an aryl group is phenyl. Substituted phenyl orsubstituted aryl denotes a phenyl group or aryl group substituted withone, two, three, four or five substituents chosen, unless otherwisespecified, from halogen (F, Cl, Br, I), hydroxy, protected hydroxy,cyano, nitro, alkyl (such as C₁-C₆ alkyl), alkoxy (such as C₁-C₆alkoxy), benzyloxy, carboxy, protected carboxy, carboxymethyl, protectedcarboxymethyl, hydroxymethyl, protected hydroxymethyl, aminomethyl,protected aminomethyl, trifluoromethyl, alkylsulfonylamino,arylsulfonylamino, heterocyclylsulfonylamino, heterocyclyl, aryl, orother groups specified. One or more methyne (CH) and/or methylene (CH₂)groups in these substituents may in turn be substituted with a similargroup as those denoted above. Examples of the term “substituted phenyl”includes but is not limited to a mono- or di(halo)phenyl group such as2-chlorophenyl, 2-bromophenyl, 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl,4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl,2-fluorophenyl and the like; a mono- or di(hydroxy)phenyl group such as4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, theprotected-hydroxy derivatives thereof and the like; a nitrophenyl groupsuch as 3- or 4-nitrophenyl; a cyanophenyl group, for example,4-cyanophenyl; a mono- or di(lower alkyl)phenyl group such as4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,4-(iso-propyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the like; amono or di(alkoxy)phenyl group, for example, 3,4-dimethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl, 3-ethoxyphenyl,4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl andthe like; 3- or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or(protected carboxy)phenyl group such 4-carboxyphenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as3-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono-or di(N-(methylsulfonylamino))phenyl such as3-(N-methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups where the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl, and the like, as well as trisubstituted phenylgroups where the substituents are different, for example3-methoxy-4-benzyloxy-6-methyl sulfonylamino,3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and tetrasubstitutedphenyl groups where the substituents are different such as3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. Particularsubstituted phenyl groups are 2-chlorophenyl, 2-aminophenyl,2-bromophenyl, 3-methoxyphenyl, 3-ethoxy-phenyl, 4-benzyloxyphenyl,4-methoxyphenyl, 3-ethoxy-4-benzyloxyphenyl, 3,4-diethoxyphenyl,3-methoxy-4-benzyloxyphenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-phenyl,3-methoxy-4-(1-chloromethyl)benzyloxy-6-methyl sulfonyl aminophenylgroups. Fused aryl rings may also be substituted with the substituentsspecified herein, for example with 1, 2 or 3 substituents, in the samemanner as substituted alkyl groups.

“Carbocyclyl”, “carbocyclylic”, “carbocycle” and “carbocyclo” alone andwhen used as a moiety in a complex group such as a carbocycloalkylgroup, refers to a mono-, bi-, or tricyclic aliphatic ring having 3 to14 carbon atoms, for example 3 to 7 carbon atoms, which may be saturatedor unsaturated, aromatic or non-aromatic. Particular saturatedcarbocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl groups. In a particular embodiment saturated carbocyclicgroups are cyclopropyl and cyclohexyl. In another particular embodimenta saturated carbocyclic group is cyclohexyl. Particular unsaturatedcarbocycles are aromatic e.g. aryl groups as previously defined. Inparticular unsaturated carbocycle is phenyl. The terms “substitutedcarbocyclyl”, “carbocycle” and “carbocyclo” mean these groupssubstituted by the same substituents as the “substituted alkyl” group.

“Carboxy-protecting group” as used herein refers to one of the esterderivatives of the carboxylic acid group commonly employed to block orprotect the carboxylic acid group while reactions are carried out onother functional groups on the compound. Examples of such carboxylicacid protecting groups include 4-nitrobenzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl,benzhydryl, 4,4′-dimethoxybenzhydryl, 2,2′,4,4′-tetramethoxybenzhydryl,alkyl such as t-butyl or t-amyl, trityl, 4-methoxytrityl,4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 2-phenylprop-2-yl,trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl,beta-(trimethylsilyl)ethyl, beta-(di(n-butyl)methylsilyl)ethyl,p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl,1-(trimethylsilylmethyl)prop-1-en-3-yl, and like moieties. The speciesof carboxy-protecting group employed is not critical so long as thederivatized carboxylic acid is stable to the condition of subsequentreaction(s) on other positions of the molecule and can be removed at theappropriate point without disrupting the remainder of the molecule. Inparticular, it is important not to subject a carboxy-protected moleculeto strong nucleophilic bases or reductive conditions employing highlyactivated metal catalysts such as Raney nickel. (Such harsh removalconditions are also to be avoided when removing amino-protecting groupsand hydroxy-protecting groups, discussed below.) Particular carboxylicacid protecting groups are the allyl and p-nitrobenzyl groups. Similarcarboxy-protecting groups used in the cephalosporin, penicillin andpeptide arts can also be used to protect a carboxy group substituents.Further examples of these groups are found in T. W. Greene and P. G. M.Wuts, “Protective Groups in Organic Synthesis”, 2^(nd) ed., John Wiley &Sons, Inc., New York, N.Y., 1991, chapter 5; E. Haslam, “ProtectiveGroups in Organic Chemistry”, J. G. W. McOmie, Ed., Plenum Press, NewYork, N.Y., 1973, Chapter 5, and T. W. Greene, “Protective Groups inOrganic Synthesis”, John Wiley and Sons, New York, N.Y., 1981, Chapter5. The term “protected carboxy” refers to a carboxy group substitutedwith one of the above carboxy-protecting groups.

“Guanidine” denotes the group —NH—C(NH)—NHR wherein R is H or alkyl oraralkyl. A particular guanidine is the group —NH—C(NH)—NH₂.

“Hydroxy-protecting group” as used herein refers to a derivative of thehydroxy group commonly employed to block or protect the hydroxy groupwhile reactions are carried out on other functional groups on thecompound. Examples of such protecting groups includetetrahydropyranyloxy, acetoxy, carbamoyloxy, trifluoro, chloro, carboxy,bromo and iodo groups. Further examples of these groups are found in T.W. Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis”,2^(nd) ed., John Wiley & Sons, Inc., New York, N.Y., 1991, chapters 2-3;E. Haslam, “Protective Groups in Organic Chemistry”, J. G. W. McOmie,Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, and T. W. Greene,“Protective Groups in Organic Synthesis”, John Wiley and Sons, New York,N.Y., 1981. The term “protected hydroxy” refers to a hydroxy groupsubstituted with one of the above hydroxy-protecting groups.

“Heterocyclic group”, “heterocyclic”, “heterocycle”, “heterocyclyl”, or“heterocyclo” alone and when used as a moiety in a complex group such asa heterocycloalkyl group, are used interchangeably and refer to anymono-, bi-, or tricyclic, saturated or unsaturated, aromatic(heteroaryl) or non-aromatic ring having the number of atoms designated,generally from 5 to about 14 ring atoms, where the ring atoms are carbonand at least one heteroatom (nitrogen, sulfur or oxygen). In aparticular embodiment the group incorporates 1 to 4 heteroatoms.Typically, a 5-membered ring has 0 to 2 double bonds and 6- or7-membered ring has 0 to 3 double bonds and the nitrogen or sulfurheteroatoms may optionally be oxidized (e.g. SO, SO₂), and any nitrogenheteroatom may optionally be quaternized. Particular non-aromaticheterocycles include morpholinyl (morpholino), pyrrolidinyl, oxiranyl,oxetanyl, tetrahydrofuranyl, 2,3-dihydrofuranyl, 2H-pyranyl,tetrahydropyranyl, thiiranyl, thietanyl, tetrahydrothietanyl,aziridinyl, azetidinyl, 1-methyl-2-pyrrolyl, piperazinyl andpiperidinyl.

A “heterocycloalkyl” group is a heterocycle group as defined abovecovalently bonded to an alkyl group as defined above. Particular5-membered heterocycles containing a sulfur or oxygen atom and one tothree nitrogen atoms include thiazolyl, such as thiazol-2-yl andthiazol-2-yl N-oxide, thiadiazolyl such as 1,3,4-thiadiazol-5-yl and1,2,4-thiadiazol-5-yl, oxazolyl such as oxazol-2-yl, and oxadiazolylsuch as 1,3,4-oxadiazol-5-yl, and 1,2,4-oxadiazol-5-yl. Particular5-membered ring heterocycles containing 2 to 4 nitrogen atoms includeimidazolyl such as imidazol-2-yl; triazolyl such as 1,3,4-triazol-5-yl,1,2,3-triazol-5-yl, and 1,2,4-triazol-5-yl, and tetrazolyl such as1H-tetrazol-5-yl. Particular benzo-fused 5-membered heterocycles arebenzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Particular6-membered heterocycles contain one to three nitrogen atoms andoptionally a sulfur or oxygen atom, for example pyridyl, such aspyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl such as pyrimid-2-yland pyrimid-4-yl; triazinyl such as 1,3,4-triazin-2-yl and1,3,5-triazin-4-yl; pyridazinyl such as pyridazin-3-yl, and pyrazinyl.Substituents for optionally substituted heterocycles, and furtherexamples of the 5- and 6-membered ring systems discussed above can befound in W. Druckheimer et al, U.S. Pat. No. 4,278,793.

“Heteroaryl” alone and when used as a moiety in a complex group such asa heteroaralkyl group, refers to any mono-, bi-, or tricyclic aromaticring system having the number of atoms designated where at least onering is a 5-, 6- or 7-membered ring containing from one to fourheteroatoms selected from the group nitrogen, oxygen, and sulfur (Lang'sHandbook of Chemistry, supra). Included in the definition are anybicyclic groups where any of the above heteroaryl rings are fused to abenzene ring. The following ring systems are examples of the heteroaryl(whether substituted or unsubstituted) groups denoted by the term“heteroaryl”: thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl,isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl,oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl, thiazinyl, oxazinyl, triazinyl,thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl,tetrazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl,dihydropyrimidyl, tetrahydropyrimidyl, tetrazolo[1,5-b]pyridazinyl andpurinyl, as well as benzo-fused derivatives, for example benzoxazolyl,benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl,benzoimidazolyl and indolyl. Particularly “heteroaryls” include;1,3-thiazol-2-yl, 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,2,4-thiadiazol-5-yl, 3-methyl-1,2,4-thiadiazol-5-yl,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl,2-hydroxy-1,3,4-triazol-5-yl, 2-carboxy-4-methyl-1,3,4-triazol-5-ylsodium salt, 2-carboxy-4-methyl-1,3,4-triazol-5-yl, 1,3-oxazol-2-yl,1,3,4-oxadiazol-5-yl, 2-methyl-1,3,4-oxadiazol-5-yl,2-(hydroxymethyl)-1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl,1,3,4-thiadiazol-5-yl, 2-thiol-1,3,4-thiadiazol-5-yl,2-(methylthio)-1,3,4-thiadiazol-5-yl, 2-amino-1,3,4-thiadiazol-5-yl,1H-tetrazol-5-yl, 1-methyl-1H-tetrazol-5-yl,1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-ylsodium salt, 1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonicacid)-1H-tetrazol-5-yl sodium salt, 2-methyl-1H-tetrazol-5-yl,1,2,3-triazol-5-yl, 1-methyl-1,2,3-triazol-5-yl,2-methyl-1,2,3-triazol-5-yl, 4-methyl-1,2,3-triazol-5-yl, pyrid-2-ylN-oxide, 6-methoxy-2-(n-oxide)-pyridaz-3-yl, 6-hydroxypyridaz-3-yl,1-methylpyrid-2-yl, 1-methylpyrid-4-yl, 2-hydroxypyrimid-4-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-6-methoxy-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-as-triazin-3-yl,2,5-dihydro-5-oxo-2-methyl-as-triazin-3-yl,2,5-dihydro-5-oxo-2,6-dimethyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl and8-aminotetrazolo[1,5-b]-pyridazin-6-yl. An alternative group of“heteroaryl” includes; 4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl,4-(carboxymethyl)-5-methyl-1,3-thiazol-2-yl sodium salt,1,3,4-triazol-5-yl, 2-methyl-1,3,4-triazol-5-yl, 1H-tetrazol-5-yl,1-methyl-1H-tetrazol-5-yl,1-(1-(dimethylamino)eth-2-yl)-1H-tetrazol-5-yl,1-(carboxymethyl)-1H-tetrazol-5-yl, 1-(carboxymethyl)-1H-tetrazol-5-ylsodium salt, 1-(methylsulfonic acid)-1H-tetrazol-5-yl, 1-(methylsulfonicacid)-1H-tetrazol-5-yl sodium salt, 1,2,3-triazol-5-yl,1,4,5,6-tetrahydro-5,6-dioxo-4-methyl-as-triazin-3-yl,1,4,5,6-tetrahydro-4-(2-formylmethyl)-5,6-dioxo-as-triazin-3-yl,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl sodium salt,2,5-dihydro-5-oxo-6-hydroxy-2-methyl-as-triazin-3-yl,tetrazolo[1,5-b]pyridazin-6-yl, and8-aminotetrazolo[1,5-b]pyridazin-6-yl.

“Inhibitor” means a compound which reduces or prevents the binding ofIAP proteins to caspase proteins or which reduces or prevents theinhibition of apoptosis by an IAP protein.

“Pharmaceutically acceptable salts” include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, carbonic acid, phosphoric acid and the like,and organic acids may be selected from aliphatic, cycloaliphatic,aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes oforganic acids such as formic acid, acetic acid, propionic acid, glycolicacid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilicacid, benzoic acid, cinnamic acid, mandelic acid, embonic acid,phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicyclic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly base addition salts are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases includes salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly organicnon-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

The present invention provides novel compounds having the generalformula I:

wherein X₁, X₂, L, R₁, R₂, R₃, R₄, R_(4′), R₅, R_(5′) and R₆ are asdefined herein.

X₁ and X₂ are each independently O or S. In a particular embodiment, X₁and X₂ are both O. In another particular embodiment X₁ and X₂ are bothS. In another particular embodiment, X₁ is S while X₂ is O. In anotherparticular embodiment, X₁ is 0 while X₂ is S.

L is a bond, —C(X₃)—, —C(X₃)NR₁₂ or —C(X₃)O— wherein X₃ is O or S andR₁₂ is H or R₁. In a particular embodiment, L is a bond. In anotherparticular embodiment, L is —C(X₃)— wherein X₃ is O. In anotherparticular embodiment, L is —C(X₃)— wherein X₃ is S. In a particularembodiment, L is —C(X₃)NH— wherein X₃ is O. In another particularembodiment, L is —C(X₃)NH— wherein X₃ is S. In a particular embodiment,L is —C(X₃)O— wherein X₃ is O. In another particular embodiment, L is—C(X₃)O— wherein X₃ is S.

R₁ is alkyl, a carbocycle, carbocycle-substituted alkyl, a heterocycleor heterocycle-substituted alkyl, wherein each is optionally substitutedwith halogen, hydroxyl, mercapto, carboxyl, alkyl, haloalkyl, alkoxy,alkylsulfonyl, amino, nitro, aryl and heteroaryl. In a particularembodiment, R₁ is alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl or heteroaralkyl wherein each is optionally substituted withhalogen, hydroxyl, mercapto, carboxyl, alkyl, haloalkyl amino, nitro,aryl and heteroaryl. In a particular embodiment R₁ is selected from thegroup consisting of formula IIa, IIb, IIc and IId:

whereinR₇ is H, alkyl, alkoxy, halogen, hydroxyl, mercapto, carboxyl, amino,nitro, aryl, aryloxy, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl,or heteroaralkyl; R₈ is H, alkyl, aryl or heteroaryl optionallysubstituted with halogen, hydroxyl, alkoxy, carboxyl, or amino; R₉ is Hor alkyl; Y is NH, NR₁₀, O or S wherein R₁₀ is H, alkyl or aryl; Z isCH, CH₂ or N; and m is 0, 1, 2 or 3. In a particular embodiment, R₁ isselected from the group consisting of formula IIa, IIb, IIc and IIdwhile L is —C(X₃)— and in particular when X₃ is O.

When R₁ is the group of formula IIa, R₇ is may be H, halogen, hydroxylor alkoxy. In a particular embodiment R₇ is H, methyl, F or methoxy. Inanother particular embodiment, R₁ is selected from the group consistingof IIa¹, IIa², IIa³ and IIa⁴:

In a particular embodiment, R₁ is selected from the group consisting ofIIa¹, IIa², IIa³ and IIa⁴ while L is —C(X₃)—. In a particular embodimentR₁ is the group of formula IIa¹. In a particular embodiment R₁ is thegroup of formula IIa2. In a particular embodiment R₁ is the group offormula IIa³. In a particular embodiment R₁ is the group of formulaIIa⁴.

When R₁ is the group of formula IIb, R₇ may be H or methyl. In aparticular embodiment R₁ is benzothiophene. In another particularembodiment, R₁ is indole. In another particular embodiment R₁ isN-methyl indole. In another particular embodiment R₁ is benzofuran. Inanother particular embodiment R₁ is 2,3-dihydro-benzofuran.

When R₁ is the group of formula IIc, m is 0 or 1; R₇ is H, alkyl orhalogen; R₈ is H, alkyl or aryl; and R₉ is H or methyl. In a particularembodiment R₁ is the group of formula IIc¹:

wherein R₇ and R_(7′) are each independently H, alkyl, alkoxy, halogen,hydroxyl, mercapto, carboxyl, amino, nitro, aryl, aryloxy, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroaralkyl; R₉ is H oralkyl; and m is 0 or 1. In a particular embodiment, R₉ is H. In anotherparticular embodiment R₉ is methyl. In another particular embodiment mis 0. In another particular embodiment m is 1. In a particularembodiment m is 0, R₇ and R_(7′) are both H and R₉ is H. In anotherparticular embodiment, m is 1, R₇ and R_(7′) are both H and R₉ is H. inanother particular embodiment, m is 0, R₇ and R_(7′) are both H and R₉is methyl. In another particular embodiment, m is 1, R₇ and R_(7′) areboth H and R₉ is methyl.

When R₁ is the group IId, R₇ is H, alkyl or aryl; m is 0 or 1; R₉ andR_(9′) are independently H or alkyl. In a particular embodiment m is 0;R₇ is H or aryl. In a particular embodiment m is O and R₇ is H. Inanother particular embodiment m is 0 and R₇ is 2-phenyl. In anotherembodiment m is 1; R₇ is H; and R₉ and R_(9′) are both H. In anotherembodiment m is 1; R₇ is H and R₉ and R_(9′) are both methyl.

When L is a bond, R₁ is may be alkyl, cycloalkylalkyl, aryl, aralkyl, aheterocycle or heterocyclylalkyl each optionally substituted withhalogen hydroxyl, mercapto, carboxyl, alkyl, haloalkyl, amino, nitro,aryl and heteroaryl. In this particular embodiment, R₁ is heteroaryloptionally substituted with aryl or heteroaryl. In a particularembodiment R₁ is

wherein Q₁ is NR₁₁, O or S; Q₂, Q₃ and Q₄ are independently CR₁₁ or N;wherein R₁₁ is H, alkyl, aryl, cycloalkyl or a heterocycle optionallysubstituted with halogen hydroxyl, mercapto, carboxyl, alkyl, haloalkyl,amino, nitro, aryl or heteroaryl. In such an embodiment, R₁₁ may be anoptionally substituted phenyl or pyridyl group. In a particularembodiment R₁₁ is

In a particular embodiment when L is —C(X₃)NR₁₂, R₁₂ is H, alkyl,cycloalkylalkyl, aryl, aralkyl, a heterocycle or heterocyclylalkyl eachoptionally substituted with halogen hydroxyl, mercapto, carboxyl, alkyl,haloalkyl, amino, nitro, aryl and heteroaryl. In this particularembodiment, R₁₂ is aryl optionally substituted with halogen, hydroxyl orhaloalkyl. In a particular embodiment R₁₂ is phenyl.

R₂ is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a heterocycleor heterocyclylalkyl. In a particular embodiment R₂ is alkyl orcycloalkyl. In an embodiment of the invention R₂ is t-butyl, isopropyl,cyclohexyl, cyclopentyl or phenyl. In a particular embodiment, R₂ iscyclohexyl. In another embodiment R₂ is tetrahydropyran-4-yl. In anotherparticular embodiment, R₂ is isopropyl (i.e. the valine amino acid sidechain). In another particular embodiment, R₂ is t-butyl. In a particularembodiment R₂ is oriented such that the amino acid, or amino acidanalogue, which it comprises is in the L-configuration.

R₃ is H or alkyl. In a particular embodiment R₃ is H or methyl, ethyl,propyl or isopropyl. In a particular embodiment R₃ is H or methyl. Inanother particular embodiment R₃ is methyl. In another particularembodiment, R₃ is t-butyl. In another particular embodiment R₃ isoriented such that the amino acid, or amino acid analogue, which itcomprises is in the L-configuration.

R₄ and R_(4′) are independently H, alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, heteroaryl, or heteroaralkyl wherein each is optionallysubstituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy,amino and nitro. In a particular embodiment R₄ and R_(4′) are both H. Inanother particular embodiment R₄ is methyl and R_(4′) is H. In aparticular embodiment, R_(4′) is H and R₄ is H, alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl or heteroaralkyl. In aparticular embodiment R₄ is a group selected from the group consistingof:

R₅ and R_(5′) are each independently H or alkyl. In a particularembodiment, R₅ and R_(5′) are H or methyl. In a particular embodiment,R₅ is H and R_(5′) is methyl. In another particular embodiment, R₅ ismethyl and R_(5′) is H. In another particular embodiment R₅ and R_(5′)are both methyl. In another particular embodiment, R₅ and R_(5′) areboth H.

R₆ is H or alkyl. In a particular embodiment, R₆ is H or methyl. In aparticular embodiment R₆ is H.

Compounds of the invention contain one or more asymmetric carbon atoms.Accordingly, the compounds may exist as diastereomers, enantiomers ormixtures thereof. The syntheses of the compounds may employ racemates,diastereomers or enantiomers as starting materials or as intermediates.Diastereomeric compounds may be separated by chromatographic orcrystallization methods. Similarly, enantiomeric mixtures may beseparated using the same techniques or others known in the art. Each ofthe asymmetric carbon atoms may be in the R or S configuration and bothof these configurations are within the scope of the invention. In aparticular embodiment, compounds of the invention have the followingstereochemical configuration of formula I′

The invention also encompasses prodrugs of the compounds describedabove. Suitable prodrugs where applicable include known amino-protectingand carboxy-protecting groups which are released, for examplehydrolyzed, to yield the parent compound under physiologic conditions. Aparticular class of prodrugs are compounds in which a nitrogen atom inan amino, amidino, aminoalkyleneamino, iminoalkyleneamino or guanidinogroup is substituted with a hydroxy (OH) group, an alkylcarbonyl (—CO—R)group, an alkoxycarbonyl (—CO—OR), an acyloxyalkyl-alkoxycarbonyl(—CO—O—R—O—CO—R) group where R is a monovalent or divalent group and asdefined above or a group having the formula —C(O)—O—CP1P2-haloalkyl,where P1 and P2 are the same or different and are H, lower alkyl, loweralkoxy, cyano, halo lower alkyl or aryl. In a particular embodiment, thenitrogen atom is one of the nitrogen atoms of the amidino group of thecompounds of the invention. These prodrug compounds are preparedreacting the compounds of the invention described above with anactivated acyl compound to bond a nitrogen atom in the compound of theinvention to the carbonyl of the activated acyl compound. Suitableactivated carbonyl compounds contain a good leaving group bonded to thecarbonyl carbon and include acyl halides, acyl amines, acyl pyridiniumsalts, acyl alkoxides, in particular acyl phenoxides such asp-nitrophenoxy acyl, dinitrophenoxy acyl, fluorophenoxy acyl, anddifluorophenoxy acyl. The reactions are generally exothermic and arecarried out in inert solvents at reduced temperatures such as −78 toabout 50 C. The reactions are usually also carried out in the presenceof an inorganic base such as potassium carbonate or sodium bicarbonate,or an organic base such as an amine, including pyridine, triethylamine,etc. One manner of preparing prodrugs is described in U.S. Ser. No.08/843,369 filed Apr. 15, 1997 (corresponding to PCT publicationWO9846576) the contents of which are incorporated herein by reference intheir entirety.

Particular compounds of formula I include the following:

SYNTHESIS

Compounds of the invention are prepared using standard organic synthetictechniques from commercially available starting materials and reagents.It will be appreciated that synthetic procedures employed in thepreparation of compounds of the invention will depend on the particularsubstituents present in a compound and that various protection anddeprotection may be required as is standard in organic synthesis. Forcompounds of the invention in which L is —C(X₃)—, a general syntheticscheme may involve an N-protected 6-amino-azabicyclo-octane group thatis coupled to an activated ester of the desired acid (e.g.naphthalene-carboxylic acid) followed by deprotection of the ring amineand subsequent coupling of amino acid residues thereto using typicalamide coupling procedures.

The N-protected 6-amino-azabicyclo-octane intermediate may be preparedaccording to the procedures described in Cary et al, TetrahedronLetters, 1989, 30:5547 illustrated in scheme 2 below. In general, anactivated ester of cyclopentene acetic acid is coupled to methylbenzylamine. The methylbenzyl group serves as an amine protecting for the ringproduct prior to coupling to amino acid residues. The resulting amide isreduced with lithium aluminum hydride to form a secondary amine which isthen reacted with N-bromosuccinimide. The resulting N-bromo amine iscyclized with a catalytic amount of cuprous bromide to generated the6-bromo substituted azabicyclo-octane ring. The ring is then reactedwith ammonium hydroxide to convert the 6-bromo group to thecorresponding 6-amino ring intermediate which then may used in thesynthesis of the compounds of the invention.

In a particular embodiment, the methyl benzyl amine is enantiomericallypure. Use of such a chiral auxiliary enables the convenient separationof the diastereomers of the azabicyclo-octane ring, for example RP-HPLCor silica gel column. Separation of the diastereomers may be performedwith the 6-bromo substituted ring or the 6-amino substituted ring priorto removal of the chiral auxiliary protecting group.

Alternatively, the compounds of the invention may be prepared accordingto the general Scheme 3, by sequential coupling of amino acids residuesincorporating R₂ and R₃ to the azabicyclo-octane ring followed bycoupling an R₄-containing acid to the 6-amino group on theazabicyclo-octane ring. In this method, the starting azabicyclo-octanering is protected at the primary 6-amino substituent, for example with aTeoc group (trimethylsilylethyloxycarbonyl) followed by deprotection ofthe secondary ring amine. Using standard peptide coupling methods, theresulting deprotected ring amine is coupled with an R₂-containingresidue and then an R₃-containing residue. The Teoc group is thenremoved with TASF (tris(dimethylamino)sulfoniumdifluorotrimethylsilicate) and the deprotected 6-amino group is coupledwith an R₄-containing acid.

For compounds of the invention in which L is —C(X₃)O—, i.e. a carbamate,a general synthetic scheme may involve reacting the N-protected6-amino-azabicyclo-octane intermediate with a chloroformate of R₁(Cl—C(O)O—R₁).

For compounds of the invention in which L is —C(X₃)NR₁₂—, i.e. a urea, ageneral synthetic scheme may involve reacting the N-protected6-amino-azabicyclo-octane intermediate withpara-nitrophenylchloroformate followed by reacting the resultingcarbamate with primary or secondary amine NR₁R₁₂ under strong basicconditions as illustrated in scheme 4.

Compounds in which L is a bond, i.e. R₁ and the nitrogen from which itdepends form an amine, may be prepared by reductive amination.Alternatively such compounds may be prepared by reacting the N-protected6-amino-azabicyclo-octane group with a methylsulfonate ester of the R₁group, for instance when R₁ is an aliphatic group such as alkyl, alkenylor alkynyl. When R₁ is aryl or heteroaryl such compounds may be preparedby reacting the N-protected 6-amino-azabicyclo-octane group with amethylsulfonyl-substituted R₁ compound according to the proceduresdescribed in Blass et, al. Bioorg. Med. Chem. Lett., 2000, 10:1543 andBakhtiar, et al J. Chem. Soc. Perkin Trans. 1. 1994, 3:239. For example,when L is a bond and R₁ is 1-phenyl-1H-imidazol-2-yl the compound may beprepared according to the following scheme 5.

in which 1-phenyl-1,3-dihydro-imidazole-2-thione is reacted with methyliodide followed by oxidation with m-chloroperoxybenzoic acid to give themethyl sulfonyl which is reacted with the N-protected6-amino-azabicyclo-octane group.

In another example, when L is a bond and R₁ is4-phenyl-[1,2,3]thiadiazol-5-yl, the compound may be prepared accordingto the following scheme 6 which are described in Masuda et. al., J.Chem. Soc. Perkin. Trans. 1, 1981, (5) 1591.

UTILITY

The compounds of the invention inhibit the binding of IAP protein e.g.XIAP and ML-IAP, in cells to caspases, e.g. caspases 3, 7 and/or 9.Accordingly, the compounds of the invention are useful for inducingapoptosis in cells or sensitizing cells to apoptotic signals, inparticular cancer cells that overexpress IAP proteins. More broadly, thecompounds can be used for the treatment of all cancer types which failto undergo apoptosis. Examples of such cancer types includeneuroblastoma, intestine carcinoma such as rectum carcinoma, coloncarcinoma, familiary adenomatous polyposis carcinoma and hereditarynon-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma,larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary glandcarcinoma, gastric carcinoma, adenocarcinoma, medullary thyroideacarcinoma, papillary thyroidea carcinoma, renal carcinoma, kidneyparenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpuscarcinoma, endometrium carcinoma, chorion carcinoma, pancreaticcarcinoma, prostate carcinoma, testis carcinoma, breast carcinoma,urinary carcinoma, melanoma, brain tumors such as glioblastoma,astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermaltumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acutelymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cellleukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma,bronchial carcinoma, small cell lung carcinoma, non-small cell lungcarcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma,choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma,osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma,Ewing sarcoma and plasmocytoma.

Compounds of the invention are useful for sensitizing cells to apoptoticsignals. Accordingly, the compounds may be administered prior to,concomitantly with, or following administration of radiation therapy orcytostatic or antineoplastic chemotherapy. Suitable cytostaticchemotherapy compounds include, but are not limited to (i)antimetabolites, such as cytarabine, fludarabine,5-fluoro-2′-deoxyuridine, gemcitabine, hydroxyurea or methotrexate; (ii)DNA-fragmenting agents, such as bleomycin, (iii) DNA-crosslinkingagents, such as chlorambucil, cisplatin, cyclophosphamide or nitrogenmustard; (iv) intercalating agents such as adriamycin (doxorubicin) ormitoxantrone; (v) protein synthesis inhibitors, such as L-asparaginase,cycloheximide, puromycin or diphtheria toxin; (Vi) topoisomerase Ipoisons, such as camptothecin or topotecan; (vii) topoisomerase IIpoisons, such as etoposide (VP-16) or teniposide; (viii)microtubule-directed agents, such as colcemid, colchicine, paclitaxel,vinblastine or vincristine; (ix) kinase inhibitors such as flavopiridol,staurosporin, STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine);(x) miscellaneous investigational agents such as thioplatin, PS-341,phenylbutyrate, ET-18-OCH₃, or farnesyl transferase inhibitors(L-739749, L-744832); polyphenols such as quercetin, resveratrol,piceatannol, epigallocatechine gallate, theaflavins, flavanols,procyanidins, betulinic acid and derivatives thereof; (xi) hormones suchas glucocorticoids or fenretinide; (xii) hormone antagonists, such astamoxifen, finasteride or LHRH antagonists. In a particular embodiment,compounds of the present invention are coadministered with a cytostaticcompound selected from the group consisting of cisplatin, doxorubicin,taxol, taxotere and mitomycin C. A particular cytostatic compound isdoxorubicin.

Another class of active compounds which can be used in the presentinvention are those which are able to sensitize for or induce apoptosisby binding to death receptors (“death receptor agonists”). Such agonistsof death receptors include death receptor ligands such as tumor necrosisfactor a (TNF-α), tumor necrosis factor β (TNF-β, lymphotoxin-α), LT-β(lymphotoxin-β), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1) ligand,TRAMP (DR3, Apo-3) ligand, DR6 ligand as well as fragments andderivatives of any of said ligands. In a particular embodiment, thedeath receptor ligand is TNF-α. In a particular embodiment the deathreceptor ligand is Apo2L/TRAIL. Furthermore, death receptors agonistscomprise agonistic antibodies to death receptors such as anti-CD95antibody, anti-TRAIL-R1 (DR4) antibody, anti-TRAIL-R2 (DR5) antibody,anti-TRAIL-R3 antibody, anti-TRAIL-R4 antibody, anti-DR6 antibody,anti-TNF-R1 antibody and anti-TRAMP (DR3) antibody as well as fragmentsand derivatives of any of said antibodies.

For the purpose of sensitizing cells for apoptosis, the compounds of thepresent invention can be also used in combination with radiationtherapy. The phrase “radiation therapy” refers to the use ofelectromagnetic or particulate radiation in the treatment of neoplasia.Radiation therapy is based on the principle that high-dose radiationdelivered to a target area will result in the death of reproducing cellsin both tumor and normal tissues. The radiation dosage regimen isgenerally defined in terms of radiation absorbed dose (rad), time andfractionation, and must be carefully defined by the oncologist. Theamount of radiation a patient receives will depend on variousconsideration but the two most important considerations are the locationof the tumor in relation to other critical structures or organs of thebody, and the extent to which the tumor has spread. Examples ofradiotherapeutic agents are provided in, but not limited to, radiationtherapy and is known in the art (Hellman, Principles of RadiationTherapy, Cancer, in Principles I and Practice of Oncology, 24875 (Devitaet al., 4th ed., vol 1, 1993). Recent advances in radiation therapyinclude three-dimensional conformal external beam radiation, intensitymodulated radiation therapy (IMRT), stereotactic radiosurgery andbrachytherapy (interstitial radiation therapy), the latter placing thesource of radiation directly into the tumor as implanted “seeds”. Thesenewer treatment modalities deliver greater doses of radiation to thetumor, which accounts for their increased effectiveness when compared tostandard external beam radiation therapy.

Ionizing radiation with beta-emitting radionuclides is considered themost useful for radiotherapeutic applications because of the moderatelinear energy transfer (LET) of the ionizing particle (electron) and itsintermediate range (typically several millimeters in tissue). Gamma raysdeliver dosage at lower levels over much greater distances. Alphaparticles represent the other extreme, they deliver very high LETdosage, but have an extremely limited range and must, therefore, be inintimate contact with the cells of the tissue to be treated. Inaddition, alpha emitters are generally heavy metals, which limits thepossible chemistry and presents undue hazards from leakage ofradionuclide from the area to be treated. Depending on the tumor to betreated all kinds of emitters are conceivable within the scope of thepresent invention.

Furthermore, the present invention encompasses types of non-ionizingradiation like e.g. ultraviolet (UV) radiation, high energy visiblelight, microwave radiation (hyperthermia therapy), infrared (IR)radiation and lasers. In a particular embodiment of the presentinvention UV radiation is applied.

The invention also includes pharmaceutical compositions or medicamentscontaining the compounds of the invention and a therapeutically inertcarrier, diluent or excipient, as well as methods of using the compoundsof the invention to prepare such compositions and medicaments.Typically, the compounds of formula I used in the methods of theinvention are formulated by mixing at ambient temperature at theappropriate pH, and at the desired degree of purity, withphysiologically acceptable carriers, i.e., carriers that are non-toxicto recipients at the dosages and concentrations employed into agalenical administration form. The pH of the formulation depends mainlyon the particular use and the concentration of compound, but may rangeanywhere from about 3 to about 8. Formulation in an acetate buffer at pH5 is a suitable embodiment. The inhibitory compound for use herein maybe sterile. The compound ordinarily will be stored as a solidcomposition, although lyophilized formulations or aqueous solutions areacceptable.

The composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“effective amount” of the compound to be administered will be governedby such considerations, and is the minimum amount necessary to inhibitIAP interaction with caspases, induce apoptosis or sensitize a malignantcell to an apoptotic signal. Such amount is preferably below the amountthat is toxic to normal cells, or the mammal as a whole.

Generally, the initial pharmaceutically effective amount of the compoundof the invention administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, for example, about 0.1 to 20 mg/kg of patientbody weight per day, with the typical initial range of compound usedbeing 0.3 to 15 mg/kg/day. Oral unit dosage forms, such as tablets andcapsules, may contain from about 25 to about 1000 mg of the compound ofthe invention.

The compound of the invention may be administered by any suitable means,including oral, topical, transdermal, parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. An example of a suitable oral dosage formis a tablet containing about 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg ofthe compound of the invention compounded with about 90-30 mg anhydrouslactose, about 5-40 mg sodium croscarmellose, about 5-30 mgpolyvinylpyrrolidone (PVP) K30, and about 1-10 mg magnesium stearate.The powdered ingredients are first mixed together and then mixed with asolution of the PVP. The resulting composition can be dried, granulated,mixed with the magnesium stearate and compressed to tablet form usingconventional equipment. An aerosol formulation can be prepared bydissolving the compound, for example 5-400 mg, of the invention in asuitable buffer solution, e.g. a phosphate buffer, adding a tonicifier,e.g. a salt such sodium chloride, if desired. The solution is typicallyfiltered, e.g. using a 0.2 micron filter, to remove impurities andcontaminants.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. Abbreviations used herein are as follows:

DIPEA: diisopropylethylamine;DMAP: 4-dimethylaminopyridine;DME: 1,2-dimethoxyethane;DMF: dimethylformamide;EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;HATU: O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate;

NBS: N-bromosuccinimide;

TASF: tris(dimethylamino)sulfonium difluorotrimethylsilicate;THF: tetrahydrofuran;

Example 1

The 2-azabicyclo[3.3.0]octane rings were prepared according to theprocedures in Corey et al, Tetrahedron Letters, 1989, 30:5547.

EDC (38.34 g, 200 mmol), DMAP (2.44 g, 20 mmol), cyclopentene aceticacid (25.0 g, 198 mmol), and R-α-methyl benzyl amine (25 mL, 196 mmol)were added sequentially to CH₂Cl₂ (500 mL). The solution was maintainedat rt. for 2 h, then washed with 1N HCl (3×100 mL), 1N NaOH (3×100 mL),brine (1×100 mL), dried (Na₂SO₄), filtered, and concentrated to afford43.7 g (97%) of amide 3 as a colorless solid.

To a solution of amide 3 (43.6 g, 190 mmol) in THF (430 mL) at 0° C. wasadded lithium aluminum hydride (200 mL of 1.0M solution in THF, 200mmol) over 2 h. The mixture was allowed to warm to rt, then heated atreflux for 36 h. The mixture was cooled to 0° C., then water (7.6 mL)was added drop wise, followed by 15% NaOH (8 mL) water (24 mL). Theresulting mixture was stirred vigorously over night, then filteredthrough a pad of Celite with THF and 1N NaOH (20 mL). The THF wasremoved under reduced pressure, and the residue diluted with CH₂Cl₂, thelayers were separated, and the organic layer concentrated to afford aquantitative yield of amine 4 as a colorless oil.

Amine 4 (950 mg, 4.4 mmol) was treated with N-Bromosuccinimide (980 mg,5.5 mmol) in hexanes (11 mL) at 0° C. for 2 h with vigorous stirring.Additional NBS (160 mg) was added and stirring continued for 1.5 h at 0°C. The mixture was filtered through a course frit and concentrated. Theresidue was dissolved in CH₂Cl₂ and treated with catalytic CuBr (˜1 mg)at 0° C. for 2.5 h. The solvent was removed under reduced pressure toprovide a 1:1 mixture of bromides 5 and 6, which was carried ondirectly.

The mixture of crude bromides 5 and 6 were dissolved in DME (14 mL) andconcentrated NH₄OH (7 mL) and heated at 60° C. in a sealed vessel for 18h. The solvents were removed under reduced pressure, and the residuedissolved in CH₂Cl₂ (50 mL) and extracted with 1N H₂SO₄ (1×25 mL). Theaq. layer was washed with CH₂Cl₂ (3×50 mL). The aq. layer was made basic(pH>11) with NaOH(s) and extracted with CH₂Cl₂ (3×50 mL). The combinedorganic phases were concentrated and the residue purified byreverse-phase HPLC(C₁₈, MeCN—H₂O, 0.1% TFA). Fractions containing theproduct were concentrated under reduced pressure until all of the MeCNwas removed, made basic with 1N NaOH (pH>11), and exhaustively extractedwith CH₂Cl₂. The combined organic phases were dried (Na₂SO₄), filteredand concentrated to provide amine 7 149 mg (29%) as a colorless oil.

Example 2

Amine 7 (140 mg, 0.61 mmol) was coupled with diphenyl acetic acid (148mg, 0.7 mmol) following the typical EDC coupling procedure to provide263 mg (88%) of amide 8 as a colorless oil.

A mixture of benzyl amine 8 (263 mg, 0.62 mmol), acetic acid (71 μL,1.24 mmol), 20% Pd(OH)₂.C (62 mg), and MeOH (6 mL) was maintained under1 atm. of H₂ for 8 h. The mixture was filtered through a pad of Celite,and concentrated. The residue was dissolved in CH₂Cl₂ (50 mL) and washedwith 1N NaOH (3×10 mL), brine (1×10 mL), dried (Na₂SO₄), filtered, andconcentrated to afford 200 mg (100%) of amine 9 as a colorless oil.

Example 3

A mixture of secondary amine 1 (166 mg, 0.61 mmol), CbzNt-butylglycine(166 mg, 0.61 mol), HATU (475 mg) DIPEA (1 mL) and DMF (3 ml) wasmaintained at RT 1 hr. The mixture was diluted with CH₂Cl₂ (50 mL) andwashed with 1N HCl (3×20 mL), dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by flash chromatography (SiO₂,35% ethyl acetate-hexanes) to afford 400 mg (>100%, excess wt. issolvent) of 2 as a colorless oil, which was carried on directly.

A mixture of the amide 2 from above, MeOH (15 ml), AcOH (0.3 ml) and 20%Pd(OH)₂.C (150 mg) was stirred vigorously under an atmosphere of H₂ for3 h. The mixture was filtered through Celite, with excess MeOH. The MeOHwas removed under reduced pressure, and the residue was dissolved inCH₂Cl₂ (50 mL) and washed with 0.5N NaOH (3×10 mL), dried (Na₂SO₄),filtered, and concentrated to afford 169 mg (72% over 2 steps) of amine3 as a colorless oil.

Amine 3 (169 mg, 0.44 mmol) was coupled with N-Boc-N-methyl alanine (108mg, 0.53 mmol) following the typical EDC coupling procedure to provide260 mg of protected amine 4 as a colorless oil, which was used with outfurther purification.

Protected amine 4 (265 mg) was treated with excess TAS-F in MeCN at 50°C. for 4 h. The reaction was diluted with CH₂Cl₂ (50 mL) and washed with0.5N NaOH (3×30 mL), dried (Na₂SO₄), filtered, and concentrated toafford 168 mg (90% over two steps) of amine 5 as a colorless oil.

Example 4 Compound 25

The secondary amine 1 (65 mg) was coupled to Cbz-cyclohexylglycine underthe above, then the Cbz group removed under hydrogenolysis to afford 73mg (74% over 2 steps) of amine 2 as a colorless oil.

Amine 2 (73 mg) was reacted as above to yield 76 mg (94% over 2 steps)of amine 3.

Amine 3 (39 mg) was coupled with diphenyl acetic acid under typical EDCcoupling conditions, then the Boc group removed under standardconditions. The residue was purified by HPLC to afford 58 mg (34%) offinal compound 4 as a colorless solid.

Example 5 Compound 1

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acid1H-indole-3-carboxylate under typical EDC coupling conditions followedby Boc group removal and HPLC purification to yield the final compound(31%).

Example 6 Compound 19

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acid1-methyl-1H-indole-3-carboxylate under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (39%).

Example 7 Compound 10

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acid1-methyl-1H-indazole-3-carboxylate under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (42%).

Example 8 Compound 17

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acidchloride, phenyl-acetyl chloride, followed by Boc removal with TFA andHPLC purification to yield the final compound (39%).

Example 9 Compound 18

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acidchloride, 2-Methyl-2-phenyl-propionyl chloride, followed by Boc removalwith TFA and HPLC purification to yield the final compound (54%).

Example 10 Compound 8

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with the commercially available acidchloride, 2,2-Diphenyl-propionyl chloride, followed by Boc removal withTFA and HPLC purification to yield the final compound (65%).

Example 11 Compound 14

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available benzoicacid under typical EDC coupling conditions followed by Boc group removaland HPLC purification to yield the final compound (16%).

Example 12 Compound 11

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablebenzofuran-3-carboxylic acid under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (32%).

Example 13 Compound 21

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablenaphthalene-1-carboxylic acid under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (28%).

Example 14 Compound 24

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available4-fluoro-naphthalene-1-carboxylic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (33%).

Example 15 Compound 6

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available4-methyl-naphthalene-1-carboxylic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (39%).

Example 16 Compound 4

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available(4-Fluoro-phenyl)-phenyl-acetic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (68%).

Example 17 Compound 3

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available2,3-dihydro-benzofuran-3-carboxylic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (39%).

Example 18 Compound 12

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially available2-methoxy-naphthalene-1-carboxylic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (52%).

Example 19 Compound 28

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablenaphthalene-1-carboxylic acid under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (41%).

Example 20 Compound 25

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablediphenyl-acetic acid under typical EDC coupling conditions followed byBoc group removal and HPLC purification to yield the final compound(33%).

Example 21 Compound 27

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablequinoline-4-carboxylic acid under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (78%).

Example 22 Compound 26

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablenaphthalene-2-carboxylic acid under typical EDC coupling conditionsfollowed by Boc group removal and HPLC purification to yield the finalcompound (79%).

Example 23 Compound 16

The Boc-protected amine intermediate prepared according to theprocedures of example 3 was coupled with commercially availablebenzo[b]thiophene-3-carboxylic acid under typical EDC couplingconditions followed by Boc group removal and HPLC purification to yieldthe final compound (49%).

Example 24

A mixture of dihydrobenzofuran 10 (160 mg, 0.9 mmol) DDQ (300 mg) andCH₂Cl₂ (11 mL) was maintained at room temp. for 2 days. The solution wasdiluted with 50% ethyl acetate-hexanes and washed with 0.5N NaOH (3×10mL), brine (1×10 mL), dried (Na₂SO₄), filtered, and concentrated toafford 150 mg of benzofuran methyl ester 11.

A mixture of ester 11 (150 mg) LiOH.H₂O (95 mg) THF (5 mL) and water(2.5 mL) was stirred vigorously for 2 days. The reaction was quenchedwith sat. aq. NH₄Cl (10 mL) and the THF removed under reduced pressure.The aq phase was extracted with CH₂Cl₂ (1×50 mL), dried (Na₂SO₄),filtered, and concentrated to afford 140 mg of benzofuran 12.

Example 25 IAP Inhibition Assays

In the following experiments was used a chimeric BIR domain referred toas MLXBIR3SG in which 11 of 110 residues correspond to those found inXIAP-BIR3, while the remainder correspond to ML-IAP-BIR. The chimericprotein MLXBIR3SG was shown to bind and inhibit caspase-9 significantlybetter than either of the native BIR domains, but bound Smac-basedpeptides and mature Smac with affinities similar to those of nativeML-IAP-BIR. The improved caspase-9 inhibition of the chimeric BIR domainMLXBIR3SG has been correlated with increased inhibition ofdoxorubicin-induced apoptosis when transfected into MCF7 cells.

MLXBIR3SG Sequence:

MGSSHHHHHHSSGLVPRGSHMLETEEEEEEGAGATLSRGPAFPGMGSEELRLASFYDWPLTA (SEQ IDNO.: 1) EVPPELLAAAGFFHTGHQDKVRCFFCYGGLQSWKRGDDPWTEHAKWFPGCQFLLRSKGQEYINNIHLTHSL

TR-FRET Peptide Binding Assay

Time-Resolved Fluorescence Resonance Energy Transfer competitionexperiments were performed on the Wallac Victor2 Multilabeled CounterReader (Perkin Elmer Life and Analytical Sciences, Inc.) according tothe procedures of Kolb et al (Journal of Biomolecular Screening, 1996,1(4):203). A reagent cocktail containing 300 nM his-tagged MLXBIR3SG;200 nM biotinylated SMAC peptide (AVPI); 5 μg/mL anti-hisallophycocyanin (XL665) (CISBio International); and 200 ng/mLstreptavidin-europium (Perkin Elmer) was prepared in reagent buffer (50mM Tris [pH 7.2], 120 mM NaCl, 0.1% bovine globulins, 5 mM DTT and 0.05%octylglucoside). (Alternatively, this cocktail can be made usingeuropium-labeled anti-His (Perkin Elmer) andstreptavidin-allophycocyanin (Perkin Elmer) at concentrations of 6.5 nMand 25 nM, respectively). The reagent cocktail was incubated at roomtemperature for 30 minutes. After incubation, the cocktail was added to1:3 serial dilutions of an antagonist compound (starting concentrationof 50 μM) in 384-well black FIA plates (Greiner Bio-One, Inc.). After a90 minute incubation at room temperature, the fluorescence was read withfilters for the excitation of europium (340 nm) and for the emissionwavelengths of europium (615 nm) and a allophycocyanin (665 nm).Antagonist data were calculated as a ratio of the emission signal ofallophycocyanin at 665 nm to that of the emission of europium at 615 nm(these ratios were multiplied by a factor of 10,000 for ease of datamanipulation). The resulting values were plotted as a function ofantagonist concentration and fit to a 4-parameter equation usingKaleidograph software (Synergy Software, Reading, Pa.). Indications ofantagonist potency were determined from the IC50 values. Compounds offormula I′ tested in this assay exhibited IC50 values of less than 200μM indicating IAP inhibitory activity.

compound IC₅₀ (μM) 3 <10 4 <10 5 <10 6 <10 9 <10 11 <10 12 <10 13 <10 14<10 16 <10 21 <10 22 <10 24 <10 25 <10 28 <10 42 <10

Fluorescence Polarization Peptide Binding Assay

Polarization experiments were performed on an Analyst HT 96-384(Molecular Devices Corp.) according to the procedure of Keating, S. M.,Marsters, J, Beresini, M., Ladner, C., Zioncheck, K., Clark, K.,Arellano, F., and Bodary., S. (2000) in Proceedings of SPIE: In VitroDiagnostic Instrumentation (Cohn, G. E., Ed.) pp 128-137, Bellingham,Wash. Samples for fluorescence polarization affinity measurements wereprepared by addition of 1:2 serial dilutions starting at a finalconcentration of 5 μM of MLXBIR3SG in polarization buffer (50 mM Tris[pH 7.2], 120 mM NaCl, 1% bovine globulins 5 mM DTT and 0.05%octylglucoside) to 5-carboxyfluorescein-conjugated AVPdi-Phe-NH₂(AVP-diPhe-FAM) at 5 nM final concentration.

The reactions were read after an incubation time of 10 minutes at roomtemperature with standard cut-off filters for the fluoresceinfluorophore (λ_(ex)=485 nm; λ_(em)=530 nm) in 96-well black HE96 plates(Molecular Devices Corp.). Fluorescence values were plotted as afunction of the protein concentration, and the IC50s were obtained byfitting the data to a 4-parameter equation using Kaleidograph software(Synergy software, Reading, Pa.). Competition experiments were performedby addition of the MLXBIR3SG at 30 nM to wells containing 5 nM of theAVP-diPhe-FAM probe as well as 1:3 serial dilutions of antagonistcompounds starting at a concentration of 300 μM in the polarizationbuffer. Samples were read after a 10-minute incubation. Fluorescencepolarization values were plotted as a function of the antagonistconcentration, and the IC₅₀ values were obtained by fitting the data toa 4-parameter equation using Kaleidograph software (Synergy software,Reading, Pa.). Inhibition constants (K_(i)) for the antagonists weredetermined from the IC₅₀ values.

compound IC₅₀ (μM) Ki (μM) 4 <1 <0.2 6 <1 <0.2 12 <1 <0.2 16 <1 <0.2 24<1 <0.2

1. A compound of formula I:

wherein X₁ and X₂ are independently O or S; L is a bond, —C(X₃)—, —C(X₃)NR₁₂ or —C(X₃)O— wherein X₃ is O or S and R₁₂ is H or R₁; R₁ is alkyl, a carbocycle, carbocycle-substituted alkyl, a heterocycle or heterocycle-substituted alkyl, wherein each is optionally substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, haloalkyl, alkoxy, alkylsulfonyl, amino, nitro, aryl and heteroaryl; R₂ is alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, a heterocycle or heterocyclylalkyl; R₃ is H or alkyl; R₄ and R_(4′) are independently H, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroaralkyl wherein each is optionally substituted with halogen, hydroxyl, mercapto, carboxyl, alkyl, alkoxy, amino and nitro; R₅, and R_(5′) are each independently H or alkyl; R₆ is H or alkyl; and salts and solvates thereof.
 2. The compound of claim 1, wherein R₃ is methyl.
 3. The compound of claim 2, wherein L is —C(X₃)— and R₁ is selected from the group consisting of IIa-IId:

wherein R₇ is H, alkyl, alkoxy, halogen, hydroxyl, mercapto, carboxyl, amino, nitro, aryl, aryloxy, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroaralkyl; R₈ is H, alkyl, aryl or heteroaryl optionally substituted with halogen, hydroxyl, alkoxy, carboxyl, or amino; R₉ and R_(9′) are independently H or alkyl; Y is NH, NR₁₀, O or S wherein R₁₀ is H, alkyl or aryl; Z is CH, CH₂ or N; and m is 0, 1, 2 or
 3. 4. The compound of claim 3, wherein R₁ is the group of formula IIa.
 5. The compound of claim 4, wherein R₇ is H, halogen, alkyl, hydroxyl or alkoxy.
 6. The compound of claim 4, wherein R₂ is alkyl or cycloalkyl.
 7. The compound of claim 4, wherein R₂ is isopropyl, t-butyl, or cyclohexyl.
 8. The compound of claim 4, wherein R₃ is methyl.
 9. The compound of claim 4, wherein R₄ is H or methyl, and R_(4′) is H.
 10. The compound of claim 4, wherein X₁, X₂ and X₃ are O and R₅, R_(5′) and R₆ are each H.
 11. The compound of claim 3, wherein R₁ is the group of formula IIb.
 12. The compound of claim 11, wherein the group of formula IIb is benzothiophene, indole, N-methyl indole, benzofuran or 2,3-dihydro-benzofuran.
 13. The compound of claim 11, wherein R₂ is alkyl or cycloalkyl.
 14. The compound of claim 11, wherein R₂ is isopropyl, t-butyl, or cyclohexyl.
 15. The compound of claim 11, wherein R₃ is methyl.
 16. The compound of claim 11, wherein R₄ is H or methyl, and R_(4′) is H.
 17. The compound of claim 11, wherein X₁, X₂ and X₃ are O and R₅, R_(5′) and R₆ are each H.
 18. The compound of claim 3, wherein R₁ is the group of formula IIc.
 19. The compound of claim 18, wherein R₁ is the group of the formula IIc¹:

wherein R₇ and R_(7′) are each independently H, alkyl, alkoxy, halogen, hydroxyl, mercapto, carboxyl, amino, nitro, aryl, aryloxy, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, or heteroaralkyl; R₉ is H or alkyl; and m is 0 or
 1. 20. The compound of claim 18, wherein R₇ is H, R₉ is H and m is
 0. 